Abstract P5-06-02: Loss of Rho GDIa Function and Tamoxifen Resistance in ERa-Positive Breast Cancer Cells

Abstract

Abstract Background: We have previously shown that increased phosphorylation of the S305 site in ERa via mutation of ERa can lead to tamoxifen (Tam) resistance. We have also discovered that shRNA knockdown of the Rho disassociation inhibitor (Rho GDIa) can render estrogen receptor (ERa)- positive breast cancer cells more aggressive via an increase in their metastatic ability. Rho GDIa is a negative regulator of the Rho family of proteins, which play an important role in the regulation of the actin cytoskeleton. While the Rho pathway is known to influence metastasis in breast and other types of cancers, it is unclear how this pathway impacts on Tam sensitivity. Since loss of Rho GDIa causes an increase in the activity of the downstream Rho proteins (Rho, Rac, and Cdc42), it is possible that increased activity of downstream Rho effectors may lead to Tam resistance through crosstalk with the ERa signaling pathway. Materials and Methods: Rho GDIa was stably silenced in MCF-7 cells using shRNA and its levels validated using Western blot analysis. Kinase assays were perfomed using GST-tagged ERa fragments to assess the effect of various kinases on ERa. ERE-luciferase reporter assays were used to look at ERa transactivation with estrogen and Tam treatment. In vitro-derived Tam-resistant cells were generated by culturing MCF-7 cells in Tam for >6 months. Results: Knockdown of Rho GDIa in ER-positive breast cancer cells resulted in Tam-resistant growth in vivo. Tam stimulated primary tumor growth in athymic nude mice. Mice injected with Rho GDIa knockdown cells exhibited metastatic lesions to the lung when treated with either estrogen or Tam. No lung tumors were detected in vector control transfected MCF-7 cells. Rhotekin pulldown assays revealed that Rho (A, B, and C), Rac1, and Cdc42 had higher activity in the Rho GDIa knockdown cells as compared to vector control cells. Immunecomplex kinase assays revealed that PAK1, a known downstream effector of the Rho pathway, exhibited increased activation in the Rho GDIa knockdown cells, and that this increased activity led to increased phosphorylation of ERa on the S305 residue. As expected, the acetylation of ERa was decreased when S305 was phosphorylated and this decrease in acetylation was dependent on phosphorylation of S305 since transfection of a plasmid containing a S305A mutation did not display decreased acetylation. ERE-luciferase assays showed that Rho GDIa knockdown cells had higher levels of estrogen and tamoxifen-induced activity in Rho GDIa knockdown cells as compared to vector control cells. Further possible effectors of Rho GDIa are being investigated as possible candidates playing roles in the crosstalk between these two pathways. Interestingly, cells which became Tam-resistant due to long term culture in the presence of Tam, also exhibited a decrease in endogenous Rho GDIa levels. Discussion: Loss of Rho GDIa in ERa-positive breast cancer cells conferred a Tam-resistant and metastatic phenotype. Cells with decreased Rho GDIa levels displayed increased phosphorylation of ERa, suggesting that there is a level of crosstalk between the Rho family of signaling entwork and the ERa signaling pathway. We hypothesize that disruption of this crosstalk may be an approach to prevent or reverse Tam resistance in patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-02.